The long term goal of the proposed project is to elucidate the mechanism of ation of antithrombin III by investigation of the chemical nature and structural relationships of the heparin binding site and protease binding site in this protein. Specifically, the objectives will be: (1) to elucidate the nature of the heparin binding site by identifying the specific lysine residues which interact with heparin. This part of the study will involve lysine modification of antithrombin III in the presence and absence of heparin using trinitrobenzenesulfonic acid and reductive methylation (HCHO/NaBH4). The specific lysine residues masked by heparin will be identified by using radioactive isotopes and analysis of cyanogen bromide and proteolytic fragments. (2) to elucidate the nature of the protease binding site by investigation of the kinetics and conformational aspects of the binding of thrombin and Factor Xa with refolded antithrombin III - an intermediate form of antithrombin III which has lost 60% heparin cofactor activity and 42% of the Alpha-helix known to be present in the protein but still possesses the capacity to bind thrombin. This part of the study will use conformation-sensitive tools such as circular dichroism, fluorescence and absorption difference spectroscopy. The first objective intends to test the validity of the heparin binding site model (peptide segment 281-289) predicted by the principal investigator. The second objective is intended to prove whether or not independent and non-interacting protease and heparin binding sites exist in antithrombin III. It is expected that the investigations proposed in this project which will utilize an altered form of antithrombin III will provide a useful model by which we can understand the complex function of antithrombin III and ultimately develop methods for physiological regulation of antithrombin III. The proposed study to identify the lysine residues essential for heparin binding is expected to provide information for better understanding of the mechanism by which antithrombin III affects blood coagulation. Among other outcomes visualized from this research is the possible identification of a conformational mechanism which might be considered a general feature of plasma protease-proteinase inhibitor interaction. It is expected that together with the details of molecular conformation revealed by various biophysical methods proposed in these studies, correlation can be made between the molecular order of antithrombin III and its biological activities.